Targeted gene transfer to corneal endothelium in vivo by ... - Nature

Gene Therapy (1998) 5, 1347–1354  1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt

Targeted gene transfer to corneal endothelium in vivo by electric pulse Y Oshima1, T Sakamoto1, I Yamanaka1, T Nishi2, T Ishibashi1 and H Inomata1 1

Department of Ophthalmology, Faculty of Medicine, Kyushu University, Fukuoka; and 2Departments of Neurosurgery, Kumamoto University, Kumamoto, Japan

A novel method of in vivo targeted gene transfer to intentionally selected areas of the corneal endothelium was developed. Plasmid DNA with the lacZ gene coding for ␤galactosidase was injected into the anterior chamber of adult Wistar rats, and eight pulses of electricity at intensities ranging from 5 to 40 V/cm were delivered for 50 ms to the cornea with a specially designed electric probe in order to determine the effect of gene transfer on the corneal endothelial cells. Gene expression was visualized by enzymatic color reaction using X-gal in enucleated eyes on days 1, 3, 7, 14 and 21 after gene transfer. The treated eyes were then photographed and the X-gal-positive areas were evaluated by an image analyzer. The ratios of the areas (X-gal-positive area/area of entire corneal endothelium × 100%) were then calculated to determine gene transfection efficiency. The expression of ␤-galactosidase was clearly detected in the cytoplasm of the corneal endo-

thelial cells as early as day 1 and lasted until day 21. The most intense gene expression was observed on days 1 and 3 (5.21% on day 1 and 6.45% on day 3). The expression of ␤-galactosidase on day 3 was most evident following delivery of 20 V electric pulses (0.09% at 5 V, 0.03% at 10 V, 6.45% at 20 V). ␤-Galactosidase expression was limited to the corneal endothelial cells in highly selected areas and no ␤-galactosidase expression was detected in any other intra- or extraocular tissues. In addition, no cell damage was apparent in the cornea and no inflammation was detected in any other intraocular tissues. Thus, low-voltage electric pulses successfully transferred the gene of interest to highly selective areas of the corneal endothelium without inducing any pathological changes. This targeted gene transfer method appears to have great potential for use in gene therapy for ocular diseases.

Keywords: gene therapy; corneal endothelium; electroporation; wound healing; lacZ; viral vector

Introduction Somatic gene therapy is defined as the transfer of new genetic material to the somatic cells of individuals with the aim of benefiting them therapeutically.1 The first approved human experiment with gene therapy was successfully performed in 1990 in patients deficient in adenosine deaminase.2 Following that, various gene therapy clinical trials have been attempted; the results, however, have not been satisfactory.3 One of the major obstacles to clinical gene therapy is the lack of optimal techniques for transferring genes to targeted tissues.4 For example, viral vectors such as adenoviral vectors can transfer genes effectively to intraocular tissues such as the corneal endothelium, retina, or trabecular meshwork cells. However, these vectors may also transfer the genes to untargeted intraocular tissues, sometimes causing mild to moderate inflammation.4–9 While nonviral vectors such as liposomes do not induce severe inflammation, their efficacy in gene transfer is low.4 Most importantly, however, neither viral nor nonviral vector methods can limit gene transfer to targeted tissues, and

Correspondence: T Sakamoto, Department of Ophthalmology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 8128582, Japan Received 26 August 1997; accepted 24 April 1998

surrounding tissues may receive unwanted genes, with unknown consequences. The corneal endothelium forms a single layer of approximately 400 000 cells, 4 to 6 mm thick, on the posterior corneal surface. This tissue is essential to the cornea, playing an important role in the maintenance of corneal transparency by dehydrating the corneal stroma.10,11 The corneal endothelium has recently been shown to be a modulator of the anterior chamber, serving to maintain the homeostasis of the anterior segment.12 Therefore, the transfection of genes to the corneal endothelium may provide an alternative approach to drug therapy for use in corneal wound healing and in various other ocular conditions, including genetic diseases and inflammation of the anterior chamber. To maintain good vision it is essential that the cornea be fully transparent, especially in the pupillary zone (central cornea).13 Even if the peripheral cornea is cloudy, an individual can maintain good vision as long as the central cornea remains transparent. Since the long-term and even short-term effects of gene transfer on corneal endothelial cells remain unknown, it is essential that the corneal endothelium in the central area is not exposed to gene transfer, which may cause unwanted side-effects. Thus, in vivo gene transfer must be strictly limited to specifically targeted tissues. To date, however, no effective targeted gene transfer method has been developed for use in ocular tissues.

Targeted gene transfer by electric pulse Y Oshima et al

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The present study demonstrates a method of transferring genes to a strictly circumscribed area of corneal endothelial cells, thereby eliminating the risk of inflammation or tissue damage.

Results Effect of electric pulse on gene transfer Since in a preliminary study we found that expression of the lacZ gene was most prominent on the third day after electroporation, the following data are from examinations performed at that point. Transfection efficiency increased as electroporation voltage increased, reaching a maximum at 40 V, 50 ms, and 8 pulses (the expression area was 0.09% at 5 V, 0.03% at 10 V, 6.45% at 20 V, and 10.1% at 40 V; P = 0.001) (Figure 1). Whole-mount preparations of the rat eyes following an anterior chamber injection of plasmid DNA and electroporation (E+P+) revealed a clear reaction with the X-gal substrate; X-gal appeared as a ring-shaped, dark-blue spot in the corneal endothelium, but it was clearly confined within the ringshaped area where the electrode was attached. There was no X-gal-positive reaction outside the area where the electric probe was attached in eyes in which voltages of 20 V or less were used (Figure 2a–c). Nor was any X-galpositive reaction seen in any other intraocular tissues (corneal epithelium, corneal stroma, conjunctiva, iris, lens epithelium, trabecular meshwork, ciliary epithelium, vitreous, retina, optic nerve, sclera or lacrimal gland). No corneal damage, such as opacity or persistent epithelial defects, was observed after electric pulse treatment at 20 V or less. High-voltage (40 V or higher) treatment induced corneal opacity after 3 days (Figure 2d). No corneal endothelium treated with plasmids alone (E−P+) or electric pulses alone (E+P−) showed an X-gal-positive reaction during the examination period. Nor did any of the rats in the negative control group show a positive reaction.

Time-course for the expression of lacZ gene Since the lowest voltage of electroporation consistent with safe and effective gene transfer was 20 V, the time course of gene expression was examined in eyes treated with 20 V electric pulses. X-gal activity was observed in the treated corneal endothelium from day 1 to day 21 (Figure 3). The level of X-gal activity in the corneal endothelium reached a maximum level on day 3 and then decreased over time. The ratio of X-gal-positive eyes was also the highest on days 1 to 3, and subsequently decreased over time (the expression area was 5.21% on day 1, 6.45% on day 3, 1.05% on day 7, 0.97% on day 10, 0.33% on day 14, and 0.46% on day 21) (Figure 3). No pathological changes were noted during the observation period. Histologic findings Light microscopic examination disclosed X-gal reactions in the targeted regions of the corneal endothelium (Figure 4a, b). No X-gal-positive reaction was detected in the untreated cornea or other intraocular tissues such as ciliary epithelial cells, the trabecular meshwork, the cells lining Schlemm’s canal, lens epithelial cells, or the retina (Figure 4a). Nor was any such activity seen in the ocular tissues in groups E−P+ and E+P−. In addition, no X-galpositive reaction was detected in any extraocular tissue (brain, lung, esophagus, stomach, liver, kidney, spleen, intestine and urinary bladder) on either day 3 or 21. Electron microscopic examination of the corneal epithelium 3 days after electroporation showed no apparent cell damage (Figure 5a), but did reveal rich and well-preserved endoplasmic reticula and cellular membranes in the corneal endothelial cells in both the treated and untreated groups (Figure 5b, c). Finally, the areas where electric pulses were delivered but in which no gene expression was present showed no pathological changes. In some eyes the gene transfer was repeated at three times weekly intervals with no histologically detectable inflammatory cell infiltration. Transfer of linear plasmid DNA The gene expression of linear-form DNA was then examined in the eyes treated with the same intensity, number, and duration of electric pulses as before (20 V, 50 ms, 8 pulses). The X-gal activity was observed only in the electroporated corneal endothelium from day 1 to day 7 (Figure 6). However, the ratio of the X-gal-positive area decreased dramatically on day 3 as compared with day 1. On day 3, the ratio of X-gal-positive area treated by a linear-form DNA with EP was significantly smaller than the ratio of X-gal-positive eyes treated by a circular-form DNA (the expression area of the linear-form plasmid was 5.20% on day 1, 0.15% on day 3, and 0.38% on day 7; the expression area of the circular-form plasmid was 5.21% on day 1, 6.45% on day 3, and 1.05% on day 7) (Figures 3 and 6). No X-gal activity or pathological changes were observed in any of the eyes treated by the linear-form DNA alone.

Figure 1 Transfection efficiency of electrical pulses at different voltages. Transfection efficiency was expressed as the ratio of the X-gal-positive area/area of the entire corneal endothelium (%). It increased in direct proportion to increases in voltage. The data are representative of the results of at least two independent experiments (n = 21). P ⬍ 0.01.

Discussion Targeted gene transfer In this study we successfully transferred the lacZ gene to corneal endothelial cells in vivo. LacZ gene expression


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a

b

c

d

Figure 2 Whole-mount of an eye 3 days after lacZ gene transfer by the electric-pulse method (a, 5 V; b, 10 V; c, 20 V; d, 40 V). (a,b,c) There is no corneal cloudiness or edema in the areas where the electroporation was delivered at 20 V or below. The blue-colored areas corresponding to the X-gal activity are seen inside the cornea (arrows). (c) The ring of blue-colored area corresponding to the X-gal activity and a circular electric probe are noted inside the cornea (arrow). (d) Forty-volt electric pulses created corneal cloudiness (arrowhead).

was detected for as long as 21 days and was limited to the treated corneal endothelium. None was detected in any other intraocular tissues. Importantly, the intracameral injection of plasmid DNA alone did not induce gene transfer to any other tissue, and the corneal endothelial cells expressing the lacZ gene were located only in the area where the electric pulse was delivered. This finding suggests that electroporation can be used successfully to deliver genes to specifically targeted ocular tissues and only to those tissues. By contrast, if genes were transferred by viral vectors in order, for example, to improve corneal endothelial wound healing, any tissue which could contact a viral vector might take up the gene unexpectedly, creating unpredictable side-effects. To take another example, if a gene coding for a secretory protein including growth factor were transferred, a newly produced growth factor might strongly stimulate the proliferation of cells by gene transfection, especially in areas adjacent to the gene-transfected tissue. Or, if a gene

coding for a non-secretory protein, such as p53 or bcl-2, were transferred, an unpredictable cell proliferation might occur in any anterior chamber tissue which can take that gene. By the same token, an unexpected proliferation of trabecular meshwork cells could cause uncontrollable glaucoma, or an improper proliferation of lens epithelium might induce cataract. Therefore gene transfer tissue must be handled with the greatest of care. Since functional changes in cells caused by the procedure cannot be fully controlled, even the safest gene transfer technique could alter the physiological function of cells. Gene transfer aimed at the cornea must avoid the central cornea, since the transfer could affect the basic function of the corneal endothelium, namely to keep the cornea clear, resulting in visual impairment. The precise targeting possible with our method would seem to make it suitable for treating corneal or anterior chamber diseases. However, use of higher voltages still


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Figure 3 Time-course of lacZ expression. LacZ expression was manifested as a blue-colored X-gal reaction product. Transfection efficiency (the expressed area/area of the entire corneal endothelium ×100%) was measured as described in Materials and methods). Transfection efficiency was most prominent on days 1 to 3.

could damage targeted cells. Ideally, the most important cells in this case, the endothelial cells of the pupillary zone, should be selectively treated. However, this is not possible with our method as it stands, and we recognize the need to overcome this major disadvantage.

Inflammatory response Our method allowed for effective gene transfer, with no apparent inflammatory response in any intraocular or extraocular tissue. This welcome result may be attributed to the use of naked plasmid DNA, without additional vectors or proteins. Plasmid DNA has been previously used in in vivo gene transfer, with no apparent inflammation related to the plasmid DNA.14,15 In addition, Nabel et al16 studying the systemic administration of plasmid DNA to transfer genes, found no pathological changes in animal tissues. Our own and these additional findings suggest that a limited amount of naked plasmid DNA probably does not harm tissues. Several other ways of transferring genes to the tissues of the anterior chamber have been attempted.5,17,18 An adenoviral vector-mediated method has been demonstrated to be effective in transferring genes to the anterior chamber, and a much broader area of corneal endothelium can be transfected by a single injection using this technique than is possible with our method.5 However, adenovirus-mediated gene transfection often induces either unwanted inflammation5 or needless pathologic ocular changes.19 Another method, retroviral vectormediated gene transfer, does not appear to induce severe inflammation. However, the supplemental proteins, such as polybrene, that must be injected with retroviral vector induce mild to moderate inflammation in in vivo ocular gene transfer. Also, transfection efficiency is not very high in the anterior eye, where few cells proliferate.20 Since even minimal inflammation can cause serious visual problems, the present method appears to be safer than previous methods in this respect. Possible mechanisms Electroporation, now widely used, was originally developed to introduce genes to cells in vitro21 and has

been demonstrated to be more effective in doing so than lipofection or the DEAE-dextran transfection method.22 However, in vivo electroporation has so far received little attention.23–25 It has largely been used only in cell suspensions, since a high-voltage electric pulse is necessary to complete efficient gene transfer in vitro, and high voltages are harmful to animal and human tissues. Zheng et al26 reported, however, that gene transfer could be accomplished efficiently using electric pulses in attached cells at lower voltages than those required for cell-suspension transfers. In their study, over 80% of adherent COS-M6 cells took up plasmid DNA and expressed lacZ genes, whereas the transfection efficiency was less than 20% when these cells were treated in suspension. Since attached cells have a higher surface:volume ratio than suspended cells, the transient pore required for DNA transfection was easily formed by low-voltage electroporation in these cells. Because of the high surface:volume ratio of the corneal endothelium in in vivo conditions, corneal endothelial cells are good targets for electroporation. Indeed, in the present study, the corneal endothelial cells in vivo effectively took up the genes at rather low-voltage parameters (20 V, 50 ms, 8 pulses), with no apparent damage to the corneal endothelium and surrounding tissue. We also found that circular plasmid DNA was more effectively transferred to the endothelial cells than linear plasmid DNA, a finding consistent with that of Nickoloff and Reynolds,27 who investigated linear plasmid transfer efficiency by electroporation in vitro. They explained that carrier DNA worked as a general competitive inhibitor of cellular nucleases and that linear molecules with large numbers of broken ends probably served as a signal for the induction of nucleases, which attacked the carrier and selected DNA.27 Buttrick et al28 also reported that closed circular DNA was far more effective than linear DNA in transfecting heart-muscle cells in vivo by the direct injection method. A similar phenomenon presumably was operative in our experiment. We detected X-gal-positive product (␤-galactosidase) in the corneal endothelium for at least 21 days after gene transfer, with a peak at 3 days. Probably plasmid DNA was taken up into the transfer endothelial cells immediately after electroporation, but its expression was not manifested by the translated product, ␤-galactosidase, until 3 days later. The transferred gene was expressed for at least 21 days. This temporal pattern of gene expression is similar to that observed by Acsadi et al,29 who documented the episomal location of plasmid DNAs in the nuclei of transfected cells following the direct injection of naked plasmid DNA into the adult rat heart muscle. Given cellular mechanisms such as DNA methylation or nuclease action, a gradual decrease in the levels of recombinant gene expression from episomal DNAs is not surprising.29,30 Also, since immunoreaction has been demonstrated to inhibit stable expression of transferred genes,29–31 the relatively small levels of inflammation induced by the present gene transfer technique might also have been a factor in the relatively long-lasting gene expression we found. However, the gene-transfected area produced by this method seems smaller than that created by simple injection of adenoviral vector into the anterior chamber.5,6 Both methods, then, may have a role in ensuring appropriate treatment.


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a

b

c

Figure 4 Light-microscopic photographs of an X-gal-reacted rat eye 3 days after gene transfer by electroporation (a, b, 20 V, 50 ms, 8 pulses; c, 40 V, 50 ms, 8 pulses). (a, b, c) The anterior segment of the eye. (a) Blue-colored staining (X-gal-positive products) is present only in a limited area of the corneal endothelium (arrow). There is no inflammatory reaction in the anterior segment, including the trabecular meshwork, iris stroma and lens epithelium. (a, b) No positive staining was found in any other area of the corneal endothelium, or in the stroma, epithelium, iris, lens or trabecular meshwork. The corneal stroma and epithelium are well preserved. (b) There is no apparent cell degeneration in the corneal endothelial cells with an Xgal-positive reaction. (c) Some inflammatory cells and endothelial cell damage are detected in an eye that received 40-V electric pulses. Co, cornea; AC, anterior chamber; I, iris; Ci, ciliary body; Re, retina; Epi, corneal epithelium; S, corneal stroma; End, corneal endothelium. Original magnification (a, ×80; b, c, ×800).

Clinical application The transient gene expression achieved with our method may be insufficient to benefit patients with diseases such as chronic corneal endothelial degeneration or chronic glaucoma. However, the transient expression of some growth factors probably would be effective, for example, in enhancing endothelial wound repair without excessive endothelial proliferation or tumor genesis. Also, in some chronic conditions, such as the recurrent anterior segment inflammation of Behcet’s disease, this gene transfer procedure could be repeatedly performed on the same patient, modulating the microenvironment of the eye in the most critical periods, ie during inflammatory attacks. Of all human organs, the eye is one of the best candidates for gene therapy. It is readily visible and treatable during cataract surgery, corneal transplantation and trabeculectomy. Improved versions of the present technique will probably allow effective gene transfer to intraocular

tissues in addition to the corneal endothelium, such as the retina or ciliary body.

Materials and methods Plasmids An expression vector for the ␤-galactosidase gene, pCH110, a mammalian expression vector of the lacZ gene with SV40 early promoter, was obtained from Pharmacia (Uppsala, Sweden). Plasmids grown in Escherichia coli host strain XLI-blue were purified by equilibrium centrifugation in CsCl-ethidium bromide gradients and suspended in TE buffer (pH. 8.0) at a concentration of 0.5 mg/ml. The plasmids were then suspended in phosphate-buffered saline (PBS) at several concentrations and pH was adjusted to 7.35. In some experiments, cDNA was cleaved from a circular to linear form by the diges-


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Figure 5 (a, c) Transmission-electron micrographs of the X-gal-reacted cornea in a rat eye 3 days after gene transfer by electroporation (20 V, 50 ms, 8 pulses). No pathological changes are observed on the corneal epithelium and endothelium (a, bar, 5 ␮m; c, bar, 2 ␮m). (b) Transmission electron micrograph of the corneal endothelium in a rat eye 3 days after gene transfer without electroporation (bar, 2 ␮m). No pathological changes are observed in the corneal epithelium or endothelium (S, stroma; D, Descemet’s membrane; End, endothelium; AC, anterior chamber).

tion of restriction enzyme BamHI (Takara, Tokyo, Japan).27

Plasmid injection and electroporation After obtaining the approval of Kyushu University, all animals were used humanely, in strict compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Declaration of Helsinki. Wistar rats weighing 200 to 250 g each were anesthetized by intraperitoneal injections of pentobarbital (1 mg/kg). The animals were then randomized into three experimental groups: those in which plasmids were injected in the presence of electric pulses (electroporation) (E+P+) (n = 69), those in which plasmids were injected in the absence of electroporation (E−P+) (n = 12), and those receiving electroporation without injection of plasmids (E+P−) (n = 12). A total of 5 ␮l balanced salt solution (BSS) containing plasmid DNA (500 ng/␮l) was injected with a 30-gauge needle into the anterior chamber at the lim-

bus. The concentration and amount of plasmid DNA injected were those found optimal in our preliminary studies (data not shown). All injections were monitored by direct visualization through an operating microscope. Immediately after injection, a circular stainless steel electrode, 0.5 mm in diameter, coated with gold (Unique Medical Imada, Tokyo, Japan) (Figure 7a, b) was placed on the surface of the cornea in each eye. A series of eight electric pulses with a pulse length of 50 ms was delivered with a standard square-wave electroporator BTX T820 (BTX, San Diego, CA, USA) in group E+P+ (with plasmids) and group E+P− (without plasmids). The eyes undergoing a sham operation were used as negative controls. Electroporation voltages ranged from 5 to 40 V. In some eyes, high-voltage electric pulses (100 V) were delivered to the cornea. Also, in six eyes, 20 V pulses were repeated at at least 1-week intervals. The temperature of the cornea was not affected by the electrical pulses (data not shown).


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Figure 6 Transfer of linear plasmid DNA. LacZ expression was manifested as a blue-colored X-gal reaction product. The transfection efficiency (the expressed area/area of the entire corneal endothelium ×100%) was measured as described in Materials and methods). The transfection efficiency, most prominent on day 1, was dramatically smaller on day 3.

X-gal staining and histology Enzymatic reactions were performed as previously described, with some modification.20 The rats were killed by an overdose of pentobarbital and enucleated 1, 3, 7, 14 and 21 days after electroporation with or without plasmid injection. The eyes were fixed with 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 1 h at 4°C, washed three times with PBS, and then reacted overnight at room temperature with 1 mg/ml X-gal (5-bromo-4chloro-3-indolyl galactopyranoside; Sigma Chemical, St Louis, MO, USA) in PBS containing 5 mm of K3Fe(CN)6, 5 mm of K4Fe(CN)6, 2 mm of MgCl2, 0.01% sodium deoxycholate, and 0.02% NP40. After the reaction, wholemount photographs were taken through an operating microscope at constant magnification. To assess the area reacted with the X-gal, the X-gal-reacted areas in the photographs were measured on a Macintosh computer using an image program written by Wayne Rasband at the US National Institutes of Health, and transfection efficiency was expressed as a ratio (X-gal-positive area/area of the entire corneal endothelium ×100%). The time-course of gene expression was also determined by the transfection efficiency as a ratio (X-gal-positive area/area of the entire corneal endothelium ×100%). For light-microscopic analysis, the eyes were fixed with 3.7% formaldehyde in PBS, dehydrated with a graded alcohol series and embedded in paraffin. The sections were cut and stained with hematoxylin and eosin, and the specimens were then observed by two masked observers (TI and IY) with no information about the specimens. The strength of the lacZ gene expression was determined by the blue-colored reaction products. Gene transfection to selected extraocular tissues (brain, lung, intestine, kidney, liver) was determined by histologic examination of rats killed on days 3 and 21. For the transmission electron microscopy (TEM) study, the eyes were post-fixed in 1% osmium tetroxide, dehydrated and embedded in epoxy resin. Thin sections were stained with uranyl acetate-lead citrate. Observations

Figure 7 (a) The gene transfer procedure; (b) the electric probe. Plasmid DNA was injected into the anterior chamber and electric pulses were then applied to rat corneas using an electric probe. Electricity was delivered through a ring-shaped electrode (arrow and arrowhead). The gap distance between them was 0.5 mm.

were made using an electron microscope (JEM-100CX; JEOL, Tokyo, Japan).

Statistical analysis All experiments were statistically analyzed using the Wilcoxon rank-sum P value. A P value of less than 0.05 was considered significant.

Acknowledgements This work was supported in part by Grants-in-Aid 09671804 and 09307040 from the Scientific Research from the Ministry of Education, Science, Culture and Sports of the Japanese Government, the Mochida Memorial Foundation for Medical and Pharmaceutical Research (Tokyo), the Japan National Society for the Prevention of Blindness (Tokyo), the Fukuoka Anti-Cancer Association (Fukuoka, Japan), the Kaibara Morikazu Medical Science Promotion Foundation (Fukuoka, Japan), and the Casio Science Promotion Foundation (Tokyo). The authors


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thank Drs Hiroki Sanui and Masao Uehara for their support and Dr Kenneth W Parker for editorial assistance.

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